Breast Cancer High-Penetrance Genes BRCA1 and BRCA2 Mutations Using Next-Generation Sequencing Among Iraqi Kurdish Women

Background BRCA1 and BRCA2 genes are the main high-penetrance genes that are responsible for most cases of inherited breast cancer. The present study aimed to detect the frequencies of inherited breast cancer caused by BRCA1 and BRCA2 genes among Kurdish breast cancer patients, including all the exome of these two genes, using next-generation sequencing (NGS). Methodology Seventy women who were diagnosed with breast cancer and registered at Nanakali Hospital in Erbil, Iraq, were included. Blood samples were collected for molecular testing (polymerase chain reaction (PCR)) targeting all exomes of BRCA1 and BRCA2 genes. All exome regions are sequenced by NGS using the Miseq system (Illumina Inc., San Diego, CA). Obtained data were visualized using Integrative Genomics Viewer (IGV 2.3 Software, Broad Institute, Cambridge, MA). Data were interpreted based on the National Center for Biotechnology Information (NCBI), Clinically Relevant Variation (ClinVar) archives, and other databases. Results Among 70 samples, more than forty-two variants have been detected, 20 on BRCA1 and 22 on BRCA2. Regarding clinical significance, six (14.28%) variants were pathogenic, four of them on the BRCA1 gene, which were: c.3607C>T, c.3544C>T, c.68_69del, and c.224_227delAAAG, and two pathogenic variants were on BRCA2 gene: c.100G>T, and c.1813delA. Also, two (4.76%) variants were conflict interpretations of pathogenicity, one (2.38%) was a variant of uncertain significant VUS, and the rest 29 (69%) variants were benign. In addition, four new variants (three in BRCA1 and one in BRCA2 gene), never previously reported, were identified. Conclusions In conclusion, analyzing the BRCA1/2 genes provide a better prediction for the risk of developing breast cancer in the future. Variant types and frequencies differ among different populations and ethnicities, the common mutations worldwide may not be prevalent in the Kurdish population. The current research findings will be useful for future screening studies of these two genes in the Kurdish population.


Introduction
Breast cancer is a type of cancer that forms in the cells and tissues of the breasts.It is the most common type of cancer among women, and it affects one in every eight to 10 women during their lifetime [1,2].Breast cancer is caused mainly by non-genetic factors, while hereditary factors contribute to 5%-10% of the cases.Genetic factors refer to the inheritance of an abnormal (mutated) form of a susceptible gene; most inherited cases of this cancer result from mutations in genes that are linked to the breast [3,4].
BRCA1/2 genes have expanded the knowledge of familial breast cancer, and BRCA genes are responsible for cell growth, division, and repair of damaged DNA.Their function is to keep the normal growth of breast, ovarian, and other cells.Altered forms of these genes cannot function normally and subsequently may lead to breast, ovarian, prostate, and colon cancer.In inherited breast cancer, these two genes are the most common causes; they may account for up to 10% of all cases [4][5][6].
Mutations in the BRCA1 gene cause early-onset hereditary breast cancers with an estimated risk of 57% to 81% and cause hereditary ovarian cancers with an estimated risk of 90% in high-incidence families of breast and ovarian cancers [7].Mutations in the BRCA2 gene increase the lifetime risk by 45%-85%, while hereditary ovarian cancers have a lower risk than breast cancer [3].Mutations in these two genes increase the risk and susceptibility of developing cancer, which is estimated to be up to 70% to 90% by the age of 70 [8,9].The frequency and types of variants on these two genes differ among different populations and ethnicities.Information about different variations of the BRCA1/2 genes has a key role in the clinical diagnosis and management of different cancers resulting from them [10].
Hereditary breast cancer can be detected through genetic testing; advances in molecular genetics testing allow the detection of the abnormal breast cancer gene.The clinical approaches for high-risk individuals and their families have changed rapidly due to the recent progress and implementation of next-generation sequencing (NGS) and multi-gene panel testing in the field of hereditary cancer [11,12].With these new techniques, the identification and diagnosis of genes associated with inherited susceptibility to breast cancer became much easier.Genetic testing plays a crucial role because early diagnosis of inherited abnormal genes related to breast cancer is important in the management of the disease [12,13].
Understanding BRCA1/2 mutations in any population provides a better risk assessment, prediction, and management of breast cancer.This cancer is the most common type of cancer in the Kurdistan region; the number of cases has been duplicated three times in the last decade [14].This study aimed to detect the frequencies of inherited breast cancer resulting from mutations in the BRCA1/2 genes (including all the exomes) among Kurdish breast cancer patients in the Erbil Governorate.

Sample collection
A total of 70 samples that were diagnosed with breast cancer and registered at Nanakali Hospital for Blood Diseases and Cancer, Erbil, Iraq, were included.The blood samples were preserved in ethylenediaminetetraacetic acid (EDTA) tubes until further analysis.All participants were given informed consent, and after achieving their agreements, they were included as samples by the Helsinki Declaration.

Genomic DNA extraction
Genomic DNAs were obtained from each participant and isolated from 200 µl blood samples using the HiPure Blood DNA Mini Kit (Magen, China) following the manufacturer's instructions.NanoDrop (Thermo Scientific, Multiskan Sky-1530, Singapore) was used for estimating the quality and quantity of the extracted DNA.The DNA samples with (A260-A320) and (A280-A320) ratios, concentrations higher than 40 ng/μl were obtained.

BRCA1 and BRCA2 protocol
Primers were used for the coding regions (exons and the boundary intronic regions) of these two genes.There were 22 primers for the amplification of the BRCA1 gene and 27 primers for the BRCA2 gene at INTERGEN Genetics and Rare Diseases Diagnosis Research & Application Center, Ankara, Turkey.
Polymerase chain reactions (PCRs) were carried out for all samples using the designed primers for the isolated DNA samples, and PCR products were checked by agarose gel electrophoresis (2%), as shown in the Appendix (see Figures 2, 3).PCRs belonging to each participant were mixed to obtain PCR pools that have all the amplicons of each participant in one tube.During the mixing, the efficiency of the amplification and amplicon length were considered; the volume of each PCR is inversely proportional to the efficiency of the reaction and directly proportional to the amplicon length that was estimated based on gel electrophoresis.The NucleoFast® 96 PCR kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) was used for the purification of the PCR pools for each participant.These purified pools were quantified and standardized to 0.2 ng/ul, as required for the step of sample preparation.NexteraXT sample preparation kit (Illumina Inc.) is used for preparing the sample for NGS that is performed by the Miseq system (Illumina Inc., San Diego, CA).

Miseq alignment and read
The BWA-mem 0.7.17 was used for the alignment of the raw reads to hg19 [15].Steps of sorting, duplicate marking, and base recalibration were carried out subsequently by Genome Analysis Toolkit 4 (GATK4) [16].
Variant Call was made using two separate algorithms, GATK UnifiedGenotyper and GATK HaplotypeCaller, which were both used to complement each other [16].Using the GATK SelectVariants option and based on strand bias, read depth, and call quality parameters, low-quality variants from both sets were eliminated [16].

Statistical analysis
BRCA1/2 variants were compared based on their clinical consequences using the Fisher exact test.Data analysis was carried out using GraphPad Prism version 9.0.0 (121) (GraphPad Software LLC, San Diego, CA).
A probability value of <0.05 was considered to indicate significance.

Ethical considerations
The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Medical Ethics Committee of Erbil Polytechnic University (Approval No. 23-0011).

Results
Among the 70 samples, more than 42 variants have been detected.Variants of intronic regions were neglected except for one variant that was not benign; finally, 42 distinct variants were included.In BRCA1, 20 variants were detected: 10 missense, three synonymous, two frameshift, and two nonsense variants were observed, plus three new variants.In BRCA2, 22 variants were detected: nine missense variants, eight synonymous, two nonsense, one frameshift, and one intronic variant, plus one new variant.
Among 42 variants, six of them were pathogenic, four of them on the BRCA1 gene, which were c.Also, twenty-nine benign variants were detected, of which thirteen were on the BRCA1 gene and sixteen were on the BRCA2 gene, as shown in the Appendix (see Tables 2, 3).Finally, four new variants were detected, three of them on the BRCA1 gene and one on the BRCA2 gene.Among 42 distinct variants, thirtysix of them were single nucleotide variants (SNVs), five of them were deletion (del), and one was duplication insertion (dup).

Discussion
The current study applied NGS to the whole exome of BRCA1/2 genes, as they contribute to most cases of hereditary breast cancer.The present study detected six pathogenic variants (8.57%) among 70 participants, four (5.71%) in the BRCA1 gene and two (2.85%) in the BRCA2 gene.Detecting more pathogenic variants in BRCA1 than BRCA2 has also been proven by previous studies.The current study applied NGS to the whole exome of BRCA1/2 genes, as they contribute are contributing to most cases of hereditary breast cancer.The present study detected six pathogenic variants (8.57%) among 70 participants: four (5.71%) in the BRCA1 gene and two (2.85%) in the BRCA2 gene.Detecting more pathogenic variants in BRCA1 than BRCA2 has been proven by previous studies also, a study carried out in Turkey by Geredeli and his colleagues detected 11 germline mutations in BRCA1 and eight in BRCA2.In Italy, Concolino and his colleagues detected 24 deleterious variants on BRCA1 and 13 on BRCA2.In Pakistan, a study carried out by Tariq and his colleagues detected seven variants on BRCA1, four pathogenic and three VUS, while on BRCA2 only three VUS detected [21][22][23].
Detecting only six PVs that are related to high-penetrance genes among 70 participants is comparable with rates found in previous studies that included other populations, and according to the standards, the percentage of breast cancer that results from mutations in high-penetrance genes usually ranges from 5% to 10% [24].It is true that our findings are within the usual range, but we should note that the present study detected four new novel mutations that, especially two of them, could be pathogenic because one of them is deleterious and the other is duplication, which according to the ENIGMA classification can be considered pathogenic.If these two variants are added to the reported pathogenicity, the percentage will rise to 11.43%, which goes above the usual range.Also, it is worth mentioning that the present study included BRCA1/2 genes only; it is true that these two genes are responsible for most breast cancer cases due to germline mutations, but we should not forget that there are other genes that contribute to this type of breast cancer, and if they are investigated, this percentage may increase more.
According to the present study, the percentage of breast cancer cases due to germline mutations somehow goes along with the normal range worldwide, but unfortunately, cases of breast cancer in Erbil city increased dramatically, only between 2013 and 2019, the number of cases increased about three times, from 675 to 1884 in 2019 [14].According to the same research, they revealed that the percentage of cases is predicted to increase during the present decade from 107.4% to 234.3% by 2030 in Erbil governorate.Based on these statistics, breast cancer is a main issue in this region.
On the BRCA2 gene, two variants of conflict interpretation of pathogenicity (c.3318C>G), (c.1909+12delT), and one variant of uncertain significance (c.6966G>T) have been detected and reported previously on the ClinVar database.Such variants are somehow problematic and cause confusing for decision making by genetic counselors.Two of them, (c.3318C>G) and (c.6966G>T), are not even yet reviewed by some databases like BRCAExchange and ENIGMA.For understanding that, it is important to know that the classification of the variants regarding their clinical significance is changeable, and they depend on the submitted research to the databases and the tools used for the analyses.In the future, artificial intelligence (AI) may be used more efficiently for making more precise decisions [25,26].
The current study reported four new mutations that were never reported on the BRCA genes in any databases before, so they can be reported as novel variants.Three novel variants have been detected on the BRCA1 gene: (c.3190A>C), (c.463dupC), and (c.981del), while on the BRCA2 gene, one novel variant has been detected: (c.3787A>G).Detection of new variants is normal because mutations of these two genes vary depending on geographical origin, population, and ethnicity, as has been proven previously [27,28].Further analysis and investigations using bioinformatics tools and family history are required to estimate the clinical significance of these novel variants.
Finally, many BRCA1/2 variants have been detected worldwide, and there is huge data regarding this issue around the world.Unfortunately, very little is known regarding these two genes among the Kurdish population.The present study is perhaps the first one carried out among Kurdish women using NGS looking for breast cancer cases due to germline mutations.It is true that this is a strong point for the current study, but we cannot compare it to previous data and research on our population.Until the time of performing this study, there were no NGS techniques in Erbil city, so we were obliged to transfer our practical work to Turkey, also, limited time, resources, and non-funding led to a small sample size.

Conclusions
Molecular screening using NGS, and bioinformatics tools provides important information about hereditary types of breast cancer.Having information for those who inherited pathogenic variants is helpful in the prediagnosis of BC among relatives and those who are at risk for getting the disease.The percentage of pathogenic variants among Kurdish women is lower compared to other populations.The prevalence and type of variants differ among different populations and ethnic groups, also, new, and novel variants could be detected among various ethnicities.Pretests and routine screening are recommended for all women, especially those over forty years of age.Further studies are needed, including a larger sample size and other related genes to breast cancer, to better understand hereditary breast cancer among Kurdish women.